The holo beta‐lactoglobulin lozenge reduces symptoms in cat allergy—Evaluation in an allergen exposure chamber and by titrated nasal allergen challenge

Abstract Background The allergists´ tool box in cat allergy management is limited. Clinical studies have shown that holo beta‐lactoglobulin (holoBLG) can restore micronutritional deficits in atopic immune cells and alleviate allergic symptoms in a completely allergen‐nonspecific manner. With this study, we aimed to provide proof of principle in cat allergy. Methods A novel challenge protocol for cat allergy in a standardized ECARF allergen exposure chamber (AEC) was developed. In an open pilot study (NCT05455749), patients with clinically relevant cat allergy were provoked with cat allergen for 120 min in the AEC before and after a 3‐month intervention phase (holoBLG lozenge 2x daily). Nasal, conjunctival, bronchial, and pruritus symptoms were scored every 10 min– constituting the total symptom score (TSS). Peak nasal inspiratory flow (PNIF) was measured every 30 min. In addition, a titrated nasal provocation test (NPT) was performed before and after the intervention. Primary endpoint was change in TSS at the end of final exposure compared to baseline. Secondary endpoints included changes in PNIF, NPT, and occurrence of late reactions up to 24 h after exposure. Results 35 patients (mean age: 40 years) completed the study. Compared to baseline, holoBLG supplementation resulted in significant improvement in median TSS of 50% (p < 0.001), as well as in median nasal flow by 20 L/min (p = 0.0035). 20% of patients reported late reactions after baseline exposure, but 0% after the final exposure. Conclusions Cat allergic patients profited from targeted micronutrition with the holoBLG lozenge. As previously seen in other allergies, holoBLG supplementation also induced immune resilience in cat allergies, resulting in significant symptom amelioration.


| BACKGROUND
In Europe and the US, cats are cohabitants in approximately every fourth household. In Germany, approximately 7% (6.2%-7.8%) of the general adult population is sensitized against cat 1

and in a
Korean study 34.6% of cat owners were sensitized to cats, which correlated with the suffering from asthma (OR = 2.88). 2 Also, an association of cat sensitization and atopic dermatitis (AD) has been reported. 3 Therefore, there remains a need to raise awareness for petrelated health risks, not only in terms of zoonotic diseases 4 but also of allergies, the latter especially relevant in families with increased atopic risk or having children with AD. 5 In reality, recommendations to families at risk to abstain from keeping cats are often rejected, 6 because cats are considered 'members of the family', 'best friend' or even as 'children' by the owners. 7 Indoor exposure in sensitized individuals leads to chronification of rhinitis, asthma, exacerbations of AD, and sleep impairment. 8 In contrast to dogs, which, likely through microbiota adaptions, are rather protective through early contact, 5,9 cats do not have a clinically relevant impact on microbial diversity or abundance, 10 and their role in allergy protection is still disputed. But not only cat owners are at risk; a large number of cat allergy sufferers have developed hypersensitivity without ever having a cat at home. The wide spread of the cat allergen in the public and its persistence and small particle size (PM 2.5-10 ) 11 providing ability to reach lower airways cause this phenomenon.
In addition to avoidance and symptomatic treatment, allergen immunotherapy (AIT) is in principle considered as the gold standard and first-line option for allergic rhinitis. However, unlike for other indications, data on cat AIT are sparse and not convincing: Colleagues sporadically report having successfully treated cat allergic patients with sublingual (SLIT) or subcutaneous (SCIT) AIT. Indeed, the efficacy of cat AIT, especially high-dose SCIT, 12 has been described in medium to severe disease, especially in Fel d 1 monosensitized patients, 13 and in patients with AD, 14 but there is a lack of controlled larger studies.
Biologicals in Fel d 1 allergy (severe allergic asthma) might sound promising, 15,16 but come with socio-economic challenges and without the potential of disease modification.
An additional approach is targeting the cat itself: washing the cat and drying with detergents, 17 as well as the application of modern air filters in houses with cats 11 provides some effect, but comes with practical limitations, and most importantly, has no sustained effect. A disruptive Swiss concept is the vaccination of the cat against Fel d 1 using a modern virus-like particle technology, in order to introduce neutralizing anti-Fel d 1 antibodies in the cat, which in return reduce the load of secreted allergen. 18 Several studies underpin the efficacy and persistence of this approach. 19 Even though no side effects were documented for the cats, this concept is hampered due to ethical considerations: what would be the benefit for the cat? Even more debatable in relation to a recent proposal creating knockout cats by CRISPR technology. 20 Thus, the "race towards a hypoallergenic cat" is still ongoing but challenging. 21 Also, another interesting allergen-specific approach was launched: Cat food containing chicken IgY against Fel d 1 expressed in yolks of eggs from chickens kept with cats. 22,23 The resistant IgY antibody is enterally absorbed and secreted into saliva where it complexes Fel d 1. [23][24][25] Its efficacy to reduce Fel d 1 in saliva and then also on cat dander was recently shown in 114 cat allergic patients. 26 Clearly, in this case, the food is harmless and non-invasive for the cats and also here no side effects for the animals were so far documented. 27 Despite all the above academic excitement, it is essential to focus on the group at risk, that is, i) atopic families with a strong desire to keep or acquire pets, and ii) individuals who have no cats at home but severe reactions when visiting cat households or having contact with a cat holder. It is well documented that atopic patients have micronutritional deficiencies in innate immune cells, 28 especially ferric iron (Fe 3+ ) complexed with siderophores, 29 vitamins, 30 and zinc. 31,32 These deficiencies especially drive regulatory cells into an inflammatory state, and negatively affect Th1 cell survival, all resulting in Th2 hypersensitivity. We have developed a novel approach based on the allergy-protective farm effect, 33 using beta-lactoglobulin (holoBLG) from cows as Trojan horse to shuttle micronutrients into atopic immune cells. 34 This approach was preclinically [34][35][36] and clinically 37 effective to correct the intracellular deficiencies, and thereby allergic symptoms.
Remarkably, we observed similar effects across nonrelated allergens -in birch but also grass pollen allergics 37 as well as in house dust mite-allergic patients 38,39 confirming the allergen non-specific nature of immunonutrition. 35 In the present study, we further stressed the concept that holoBLG with its micronutrients could ignite regulatory immune mechanisms in an allergen-nonspecific manner even in the highly specific cat allergies. We established a protocol for cat allergy in the standardized ECARF (ECARF Institute GmbH, Berlin, Germany) allergen exposure chamber (AEC) and supplemented patients with allergic rhinitis due to a cat with the holoBLG lozenge in this pilot study. Patients were examined before and after a 3-months supplementation of the holoBLG lozenge.
Our pilot study suggests that supplementation with the holoBLG lozenge harnessing the power of immunonutrition 31 represents a novel tool for the management of cat allergy.

| Study design
This was an open-label pilot study conducted at ECARF Institute GmbH, Berlin, Germany for clinical proof-of-concept of a holoBLG lozenge for allergen-nonspecific targeted micronutrition in cat allergic patients.
The holoBLG lozenge used in this study (immunoBON®, manufacturer Biomedical Int. R + D GmbH, Vienna, Austria, marketed by Bencard Allergie GmbH, Munich, Germany) contains the whey protein beta-lactoglobulin (BLG) combined with micronutrients: iron complexed with catechins from cocoa extract, Vitamin A, and zinc. All previous proof-of-concept studies, 30,34 preclinical studies 35,36 and clinical studies [37][38][39] were conducted with exactly this formula. In adults, the lozenge is taken twice daily over a period of 3 months.
Between October 2021 and March 2022, cat allergic patients who met the eligibility criteria at screening (visit 0, V0) were exposed twice (V2, V5) to cat dander material in the AEC of the ECARF Institute. Patients were called (V3, V6) approximately 24 h after each provocation to assess any late phase reactions. After V2, all patients were provided with the holoBLG lozenge and were instructed to slowly suck one lozenge twice daily for 3 months.
Before and after the intervention phase, capillary blood was drawn from the patients for IgE determination and a titrated nasal provocation test (NPT) was performed. The study design is shown in Supplemental Figure S1.
Measures due to the Covid-19 pandemic: All subjects had been tested negative by a corona rapid test on the days of the examination. The entire staff working on the study (technician, study nurse, doctor, subjects care) were also tested negative. To comply with local legislation, only 2 to 3 subjects could be provoked at the same time in the chamber with a sufficient distance.

| Study population
All participants received detailed information from the supervising physician and provided their written informed consent to participate at the screening visit (V0). They also agreed to the processing and storage of their data in accordance with the General Data Protection Regulation.
The study was conducted in accordance with the Declaration of Helsinki and in compliance with all federal, regional and local requirements. All data provided were pseudonymized to protect the privacy of the patients who participated in the study as mandated by the applicable laws and regulations.
After signing the informed consent form, subjects (18-65 years) were screened for eligibility. The eligibility criteria were cat allergy with rhinoconjunctivitis symptoms for ≥1 year according to the ARIA guidelines. 40 Subjects answered "Yes, moderately" or "Yes, severely" to at least two symptoms of rhinoconjunctivitis, such as runny nose, stuffy nose, itchy nose, sneezing, and itchy eyes at V0. Only patients with a wheal-size of ≥3 mm in the cat skin prick test, a positive response in NPT to cat extract and a minimum total symptom score (TSS) > 3 during the first provocation in the AEC were included.
The main exclusion criteria were sublingual or subcutaneous AIT (SLIT/SCIT) to cat allergen during the last 2 years prior screening, clinically relevant hypersensitivity to ingredients of the holoBLG lozenge, allergy to cow's milk protein, severe or uncontrolled asthma during 3 months before screening, prebronchodilator FEV1<70% before allergen exposure, relevant infectious or severe chronic diseases or contraindication to adrenaline and/or other rescue medication, simultaneous intake of anti-allergic medication prior to screening process and exposure in the AEC and supplementation with the holoBLG lozenge in the past. Wash-out times for different medications before V0, V1, V2, V4 and V5 were as follows: 3 weeks for systemic corticosteroids, 2 weeks for topical nasal corticosteroids, 7 days for cromones, 72 h for antihistamines, 3 months for antibiotics, and 1 month for pro-, pre-and synbiotics.

| Titrated nasal provocation test (NPT)
A standardized titrated NPT was performed with a cat allergen extract (cat epithelia: LETI Pharma GmbH, Witten, Germany) in three concentrations (1:100, 1:50, 1:10), starting with a dilution of 1:100 in both nostrils following a negative provocation with saline. The outcome was assessed according to current guidelines 41 : when symptoms (nasal and other symptoms) scored ≥2 points and a decrease in peak nasal inspiratory flow (PNIF) of >20% or a decrease in PNIF of >40% occurred after 15-20 min, the test was considered positive. If not, the next higher dose was used until a positive result was documented.

| Allergen exposure chamber (AEC)
The ECARF AEC is a mobile flexible chamber made of two connected standard 24 feet (7.32 m) high-cube-containers. 42,43 In this standardized and validated chamber, the exposure was performed using cat allergen (cat dander, defatted powered allergen, Greer Laboratories, Lenoir, N.C. 28645, USA.). All tests in the chamber were carried out under standardized conditions at 21°C and 55% relative humidity. During the 120-min exposure at V2 and V5, the average allergen concentration in the air breathed by each seated study patient was 400 μg/m³ of cat allergen.

| Outcome parameters
The nasal, conjunctival, bronchial and pruritus symptoms triggered in the AEC were evaluated by the patients every 10 min on a scale from 0 to 3 (no-, mild-, moderate, or severe symptoms) and summed up to constitute a total nasal symptom score (TNSS: runny-, itchy-, and BERGMANN ET AL. -3 of 10 blocked nose, sneezing), total eye symptom score (TESS: itchy-, watery eyes, gritty feeling and scoring of eye redness by the study nurse), total bronchial symptom score (TBSS: breathlessness, wheezing, cough, and asthma) and total other symptom score (TOSS/ pruritus: itchy skin, and itchy palate). TSS was defined as the sum of TNSS, TESS, TBSS, and TOSS, revealing a maximum score of 42. The primary endpoint was the change in median TSS at 120 min exposure to cat allergen in the AEC at visit V5 compared to visit V2. Secondary endpoints were the exploratory analysis of the temporal evolution of TNSS, TESS, TBSS, TOSS and TSS during each 120 min exposure and the differences between these temporal trends between V2 and V5.
Further, patients rated their personal well-being by placing a vertical line on a ten-cm line (VAS, visual analog scale) ranging in severity from "very good" (0 cm) to "very bad" (10 cm) before, every

| Statistical analysis
This open-label study was planned with 35 patients. The primary endpoint was the change in median TSS at 120 min exposure to cat allergen in the AEC at visit V5 compared to visit V2 and was analyzed employing the paired Wilcoxon test (Wilcoxon signed rank test with continuity correction), secondary endpoints were analyzed in an exploratory way.
Percent changes between AEC visits were calculated by first calculating the median of values measured during V2 and V5 separately over all patients at 120 min according to the following equa- The changes in NPT were analyzed by intra-individually comparing the concentration level needed to get a positive test at visits 1 and 4 for each subject. A test for symmetry was applied.

| Demographics
A total of 72 subjects with at least a 1-year history of moderate to severe allergic rhinoconjunctivitis with or without asthma caused by cat allergens were screened. Of these, 42 patients were included in the study; the remaining 30 subjects did not meet the inclusion criteria or could not be included due to the exclusion criteria.
After seven dropouts during the present study, 35 participants aged 24-65 years (7 male, 28 female) were able to complete the study and were included in the final data analysis (Table 1). Reasons for study dropout were quarantine due to Covid-19 infection, illnesses in the family, or further unknown reasons. All 35 patients who completed the study reported taking the lozenge as recommended. None of the subjects ever received allergen-specific immunotherapy with cat allergen. Of the 35 analyzed patients, only five had a cat at home.

| Evaluation of symptoms, personal well-being, PNIF and PEF in the AEC
The distribution of the primary endpoint (change in TSS) at the end of the final exposure (V5) compared to baseline (V2) is displayed in Figure 1A. No relevant differences were measured for PEF, as well as for the spirometry parameters (data not shown).

| Nasal provocation
Prior to and after the intervention phase a titrated NPT was per-

| DISCUSSION
The management of cat allergic patients is hampered by the lack of convincing studies for the efficacy of cat-specific allergen immunotherapy by non-compliance for allergen avoidance due to strong It has been demonstrated that holoBLG selectively nourishes regulatory immune cells with micronutrients, thereby fostering immune resilience and tolerance. 34 BLG is a lipocalin, like many of the major allergens, but acts as a tolerogen in context with its ligands   39 We are tempted to speculate that the allergen-nonspecific immune memory induced by holoBLG also provides long-term symptom relief for cat allergics.

| CONCLUSION
The results of our study suggest that the management of feline allergic rhinoconjunctivitis by targeted micronutrition with the hol-oBLG lozenge is feasible, safe and effective. Since the intervention did not include any specific cat allergen, we extrapolated that innate immune mechanisms are underlying its efficacy. In line with previous studies, the allergen-nonspecific protective farm effect of the hol-oBLG lozenge can therefore be extended to cat allergy. This study thus offers a novel opportunity in the allergist's tool box for improving the quality of life of cat allergic patients.

ACKNOWLEDGMENTS
In memory of our dear colleague and co-author, Thomas Zwingers, who passed away unexpectedly. Thomas' statistical analyses were a great contribution to the current work. His expertise, dedication and down-to-earth nature will be missed dearly. The study was funded by Bencard Allergie GmbH, Germany. The holoBLG lozenges were provided by Biomedical International R+D GmbH, Vienna, Austria. The authors thank Alan Bullimore for proofreading the manuscript.

DATA AVAILABILITY STATEMENT
The datasets generated and/or analyzed during the current study are not publicly available. Bencard Allergie GmbH is committed to providing qualified external researchers with access to pseudonymized patient data and supporting clinical documents from related studies. These requests are reviewed and approved by an independent review panel on the basis of scientific merit.

CONSENT FOR PUBLICATION
The authors provided their consent for the publication of the study results.

TRIAL REGISTRATION
The study protocol approved by the Ethics Committee of the Charité Berlin, Germany (EA1/119/21) was retrospectively registered at clinicaltrials.gov (NCT05455749).